Massively parallel screen uncovers many rare 3′ UTR variants regulating mRNA abundance of cancer driver genes

Understanding the function of rare non-coding variants represents a significant challenge. Using MapUTR, a screening method, we studied the function of rare 3′ UTR variants affecting mRNA abundance post-transcriptionally. Among 17,301 rare gnomAD variants, an average of 24.5% were functional, with 70% in cancer-related genes, many in critical cancer pathways. This observation motivated an interrogation of 11,929 somatic mutations, uncovering 3928 (33%) functional mutations in 155 cancer driver genes. Functional MapUTR variants were enriched in microRNA- or protein-binding sites and may underlie outlier gene expression in tumors. Further, we introduce untranslated tumor mutational burden (uTMB), a metric reflecting the amount of somatic functional MapUTR variants of a tumor and show its potential in predicting patient survival. Through prime editing, we characterized three variants in cancer-relevant genes (MFN2, FOSL2, and IRAK1), demonstrating their cancer-driving potential. Our study elucidates the function of tens of thousands of non-coding variants, nominates non-coding cancer driver mutations, and demonstrates their potential contributions to cancer.

understood this study or has given it a serious read.In the original comments, this reviewer their comments, but the study focused misunderstandings.First, they thought this study was testing a subset of variants already tested by Zhao et al 2014 (ref 25).This is false because these two studies are totally described it clearly in the main text.Another misunderstanding is that the reviewer thought g tumorigenesis, which is not what the author either way).
Reviewer #2 (Remarks to the authors) The author has made several improvements in this revision.However, the reviewer feels that it still does not sufficiently address the concerns previously raised.This study focused Nature Biotechnology, 2014) using a massive parallel reporter assay.Therefore, compared to the 17,312 mutations selected by Zhao et al., those demonstrated as functional in this study should have been a more concentrated representation of various important genomic functions.
Again, the reviewer misunderstood the design of this study.The variants tested here are rare variants in gnomad, unrelated to Zhao et al 2014.
> Across the 2,251 rare MapUTR functional variants that exist in at least 1 GTEx individual, 352 showed outlier expression and consistent direction of change as detected by the MapUTR assay.
Firstly, there is no description of the total number of RNA-seq in GTEx.While the criteria for outliers are not overly lenient, the figure of 352 out of 2,251 does not seem particularly high when looking across various tissues.The author should have demonstrated that the .
with GTEx data is that the genetic background is very heterogeneous.The individual with a particular variant that affect the same gene.The control, which is everyone else with the reference allele in human data but too much heterogeneity and very complicated.To get a robust signal, one will need a much larger cohort with hundreds of individuals carrying the rare allele, which > The percentage of all tested gnomAD rare variants in cancer-related genes is 71.1% (12,312 out of 17,312).We did not observe an enrichment of functional gnomAD variants in cancer-This too seems quite weak.The reviewer is concerned that this may indicate that the current experiment has not sufficiently concentrated on the important elements compared to the original data (Zhao et al.).
Again this study has nothing to do with Zhao et al.As the authors noted, the high percentage of rare gnomAD variants in cancer related genes is interesting but does not diminish the significance of this study.Those not identified to be functional in this study could be functional via mechanisms other than post-transcriptional regulation.> Among the cancer driver genes we tested, 56 of the 62 (90%) tumor suppressor genes had at least one variant that attenuates mRNA abundance, while 43 of the 50 (86%) oncogenes harbored at least one variant that enhances mRNA abundance.
The reviewer thinks this approach is flawed.They assume that the 62 tumor suppressors have numerous MapUTR variants, so it's possible that a single variant might be downregulated by chance, without leading to overall down-regulation.It was necessary to show that the MapUTRs associated with tumor suppressor genes are downregulated as a whole.
pact on mRNA abundance, rather than driving tumorigenesis as the reviewer apparently has in mind.Some of these variants could inhibit tumor growth by downregulating oncogenes or upregulating tumor suppressor genes.

Fig 5a simply
shows that some variants result in higher expression compared to reference -express these tumor suppressor genes.
In each gene, there are specific types of cancer that are associated.For example, APC is specifically seen in colorectal cancer.This perspective is completely lacking in the paper.It is inconceivable to demonstrate an example of over-expression of APC, which is thought to be inactivated in cancer, in the context of prostate cancer, especially since it is a tumor suppressor.
higher expression of APC.It has nothing to do with whether APC is overexpressed in cancer or not.
Despite preparing a list of cancer-relevant genes, MFN2, FOSL2, and IPAK1 highlighted in Fig 6 are not on this list.Nevertheless, these are being presented as mutations in cancer driver genes.Then claiming in the Abstract, "We characterized three variants in cancer-relevant genes (MFN2, FOSL2, and IPAK1)," seems to lack scientific integrity.
these 3 genes as cancer relevant genes, especially after the experiments in this study.Even if these genes are not in some list, why is this a scientific integrity issue?